Methods and compositions for prime editing nucleotide sequences

Abstract

Compositions and methods are provided herein for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis. The compositions include fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap which is synthesized by the polymerase of the fusion protein and which becomes incorporated into the target DNA molecule.

What is claimed is: 
     
       1. A prime editing guide RNA (PEgRNA) comprising:
 a) a spacer sequence; 
 b) a gRNA core that is capable of complexing with a nucleic acid programmable DNA binding protein (napDNAbp) configured to generate a cut site in a first strand of a double-stranded DNA sequence; and 
 c) an RNA extension arm comprising i) a DNA synthesis template comprising an edit template that encodes one or more edits compared to a region downstream of the cut site in the first strand of the double-stranded DNA sequence, and ii) a primer binding site that is complementary to a region upstream of the cut site in the first strand of the double-stranded DNA sequence. 
 
     
     
       2. The PEgRNA of  claim 1 , wherein the spacer sequence comprises a region of complementarity to a second strand of the double-stranded DNA sequence, and wherein the second strand is complementary to the first strand. 
     
     
       3. The PEgRNA of  claim 1 , wherein the region upstream of the cut site to which the primer binding site is complementary is immediately 5′ of the cut site. 
     
     
       4. The PEgRNA of  claim 1 , wherein the cut site is three nucleotides upstream of a protospacer adjacent motif (PAM). 
     
     
       5. The PEgRNA of  claim 1 , wherein the spacer sequence is 20 nucleotides in length. 
     
     
       6. The PEgRNA of  claim 5 , wherein the primer binding site comprises the reverse complement of nucleotides p to 17 of the spacer sequence, wherein p is an integer from 1 to 13. 
     
     
       7. The PEgRNA of  claim 1 , wherein the primer binding site is from 7 to 17 nucleotides in length. 
     
     
       8. The PEgRNA of  claim 1 , wherein the primer binding site is from 8 to 15 nucleotides in length. 
     
     
       9. The PEgRNA of  claim 1 , wherein the DNA synthesis template further comprises a homology arm that comprises a region of complementarity to a region downstream of the cut site in the first strand of the double-stranded DNA sequence. 
     
     
       10. The PEgRNA of  claim 9 , wherein the homology arm is located 5′ of the edit template. 
     
     
       11. The PEgRNA of  claim 10 , wherein the homology arm is 5 to 20 nucleotides in length. 
     
     
       12. The PEgRNA of  claim 1 , wherein the one or more edits comprise one or more nucleotide insertions, one or more nucleotide substitutions, one or more nucleotide deletions, or a combination thereof. 
     
     
       13. The PEgRNA of  claim 9 , wherein the RNA extension arm comprises, from 5′ to 3′, the homology arm, the edit template, and the primer binding site. 
     
     
       14. The PEgRNA of  claim 13 , wherein the homology arm, the edit template, and the primer binding site are directly adjacent to each other. 
     
     
       15. The PEgRNA of  claim 1 , wherein the DNA synthesis template is from 5 to 58 nucleotides in length. 
     
     
       16. The PEgRNA of  claim 1 , wherein the DNA synthesis template is from 10 to 16 nucleotides in length. 
     
     
       17. The PEgRNA of  claim 1 , wherein the DNA synthesis template is from 12 to 17 nucleotides in length. 
     
     
       18. The PEgRNA of  claim 1 , wherein the DNA synthesis template is less than 15 nucleotides in length. 
     
     
       19. The PEgRNA of  claim 1 , wherein at least one of the edits encoded by the DNA synthesis template disrupts an endogenous PAM site associated with the spacer sequence. 
     
     
       20. The PEgRNA of  claim 1 , wherein the PEgRNA is a single molecule comprising the spacer sequence, the gRNA core, and the RNA extension arm. 
     
     
       21. The PEgRNA of  claim 20 , comprising in a 5′ to 3′ orientation: the spacer sequence, the gRNA core, and the RNA extension arm. 
     
     
       22. The PEgRNA of  claim 20 , wherein the RNA extension arm is flanked by a 5′ fragment of the gRNA core and a 3′ fragment of the gRNA core. 
     
     
       23. The PEgRNA of  claim 1 , comprising at least one of a modified nucleobase, a modified sugar, a modified phosphate group, or a nucleoside analog. 
     
     
       24. The PEgRNA of  claim 1 , wherein the PEgRNA comprises one or more structures selected from the group consisting of linkers, stem loops, hairpins, toeloops, tetraloops, aptamers, and RNA-protein recruitment domains. 
     
     
       25. The PEgRNA of  claim 24 , wherein the PEgRNA comprises the one or more structures at the 3′ end of the PEgRNA. 
     
     
       26. The PEgRNA of  claim 1 , wherein the PEgRNA comprises an aptamer capable of recruiting an effector domain. 
     
     
       27. The PEgRNA of  claim 1 , wherein the nucleic acid programmable DNA binding protein is a CRISPR-Cas effector protein. 
     
     
       28. The PEgRNA of  claim 1 , wherein the nucleic acid programmable DNA binding protein comprises an HNH domain, a RuvC domain, or both. 
     
     
       29. The PEgRNA of  claim 1 , wherein the nucleic acid programmable DNA binding protein is a Cas9 protein. 
     
     
       30. The PEgRNA of  claim 26 , wherein the aptamer is a MS2 aptamer.